Sequencing analysis of the isolates of a recently identified pathogen associated with respiratory infections, human bocavirus (HBoV), allowed for identification of two virus genotypes of the virus. In the current article a new method for a simple and fast differentiation of HBoV genotypes in clinical materials is described. The test includes an amplification of a 309 bp fragment of VP1/VP2 gene of HBoV from nasopharyngeal aspirates with a subsequent incubation of a PCR mix with the BstAPI endonuclease. Upon such a digestion, the DNA fragment derived from the genotype I HBoV isolates forms two fragments of 150 and 159 bp, while that obtained from genotype 2 isolates remains unrestricted. The developed technique may be used in epidemiological studies of HBoV infection and analysis of the potential differences in biological characteristics of HboV genotypes.